The present invention relates to a process for producing a pattern of hybridization results that can be used to identify and distinguish human, other animal, or plant individuals. Traditionally, this type of identification is called "Nucleic Acid (DNA) Fingerprinting" or "DNA Profiling" because the uniqueness of nucleic acid banding patterns is conceptually similar to human fingerprints. The current method of DNA Fingerprinting requires the use of Southern Blotting. The method, according to the invention, is simple to perform, requires no interpretation, and provides results which convert to a simple bar code for computer storage and retrieval.
The Southern Blot
Nucleic acid hybridization has been employed as a common methodology for greater than 25 years. The current method of DNA Fingerprinting uses a modern Molecular Biology hybridization technique known as Southern Blotting.sup.1. The Southern blot has been a mainstay of molecular biologists since its invention in 1975. In the Southern blot method, DNA molecules must first be isolated from other cellular components by one of a variety of extraction methods. After extraction, the somewhat pure DNA is then cut with one or more restriction enzymes, generating a specific set of DNA fragments. Following DNA cutting, the fragmented DNA is separated by electrophoresis in an agarose gel. This standard technique resolves DNA fragments based on their size (molecular weight) and has greater differentiating power than other sizing procedures. Large molecules cannot pass through the matrix under the force of an electric field as readily as small molecules. Therefore, large molecules migrate more slowly. The gel resolves the DNA fragments into zones or bands of unique size.
However, the agarose gel does not provide a stable support for the subsequent hybridization reaction. Consequently, the DNA is transferred from the gel to a solid support membrane, usually composed of nylon or nitrocellulose. The DNA is transferred by either capillary action or vacuum force onto the membrane where it binds to the surface. The DNA is permanently fixed to the membrane by drying or ultraviolet light cross-linking.
The membrane is prehybridized in a special solution to block any non-specific binding sites. The membrane is then placed into a solution containing labeled, denatured probe and incubated to permit hybridization to the complementary target molecules. Many factors affect the rate, extent, and specificity of the hybridization reaction.
Hybridization is the result of complementary base pairing of single stranded nucleic acids. This binding results in formation of highly specific double stranded nucleic acid. One of the single strands typically is labeled such that it can later be measured or otherwise detected as part of a new hybrid. The hybrid molecules are generally separated from residual single stranded material, so that the hybridized label can be detected in the absence of unhybridized label. After the reaction, excess probe is washed away by a series of washing steps in detergent and dilute salt solutions.
If the probe was radioactively labeled with .sup.32 p, then autoradiography may be used to locate the position of any DNA band to which the probe bound. The membrane is simply placed next to a piece of X-ray film for several minutes to several days and then the film is developed. Wherever the radioactive particles strike the film, a dark region is formed and the target DNA can be identified. The probe may also be labeled, non-isotopically, and can be located using the appropriate detection methodology, such as fluorescence spectrophotometry, colorimetry and chemiluminescence detection.
By Southern blotting, the size of the exact fragment which bound the labeled probe is pinpointed. The Southern technique gave rise to high resolution hybridization analysis, and has formed the basis of conventional DNA Fingerprinting techniques.
Conventional DNA Fingerprinting
A conventional methodology used to show a fingerprint uses probes exhibiting restriction enzyme fragment length polymorphism (RFLP).sup.2,3 or allelic variation.sup.2-4, by Southern Blot technology. These conventional polymorphic probes have target DNA fragments, which vary in length after being cut with restriction enzymes.sup.2,3. These polymorphic probes are either restriction fragments or minisatellites. The present invention uses probes directed at nucleic acid sequences whose presence varies in the population, referred to hereinafter as Multiple Presence Polymorphisms (MPP).
In 1985, Jeffreys.sup.2 reported that simple tandem-repetitive regions of DNA, also known as minisatellites, showed tremendous fragment length polymorphism between human individuals. Jeffreys proposed that this probe, in combination with several restriction enzyme sets, could be used to produce a stable pattern of DNA bands unique to each individual tested. Furthermore, he proposed that the unique patterns could be applied to problems of human identification and determination of paternity.
The current method of DNA Fingerprinting using Southern Blotting has the following problems associated with it:
1. Time-consuming--requires one week or more to complete. PA1 2. Difficult to perform--more than one hundred steps. PA1 3. Requires highly skilled and knowledgeable technicians. PA1 4. Not standardized--variable results from test to test and from lab to lab. PA1 5. Inaccurate measurements--results require measurements of DNA fragment length, which varies with the particular electrophoresis conditions, temperature of buffer and environment, type of agarose gel used. Length measurement is also a highly subjective determination of the position of the DNA band.
Therefore, there is a definite need for a diagnostic or detection technique that is easy to perform, relatively fast, does not require significant expertise, and requires no subjectivity of interpretation. The present invention solves these needs by consisting of relatively few steps with only routine laboratory skills required, by being easily automatable through modification of instrumentation currently available on the market, and by providing results in about one day. Also, the results are measured in standard laboratory readers, such as an ELISA plate reader, spectrophotometer, fluorescence photometer or similar instrument. The results do not require any user interpretation; the output measurement converts to a numerical 1 (positive) or 0 (negative).